Total lipids were extracted as described in (Singh et al., 2011 ). In brief, single colony from WT, Δsmt1, Δsmt1+SMT1, Δgcs1 and Δgcs1+GCS1 were picked from freshly streaked YPD plates and grown in 15 ml YPD at 30°C for 20 hrs at 250 rpm. The cells were washed twice with sterile Phosphate Buffered Saline (PBS) and counted. Then, 5 × 108 cells were placed in a single glass tube, washed once with sterile DDW, and Mandala extraction was performed for extraction of inositol-containing phospholipids and phosphatidylcholine. Bligh and Dyer lipid extraction (Bligh et al., 1959 ) of neutral lipids was performed on the dried lipids obtained after Mandala lipid extraction (Mandala et al., 1995 (link)). A quarter of the samples were aliquoted for inorganic phosphate determination. The tubes were vacuum dried and used for mass spectrometry (MS) analysis. The MS and MS/MS scans were conducted using a Thermo Finnigan TSQ7000 triple quadruple mass spectrometer with electrospray ionization (Bielawski et al., 2009 (link)). The analyses included multiple reaction monitoring (MRM) for the characteristic product ions of m/z=276.2 (α-OH-Δ4-Δ8,9methyl-GlcCer at m/z 756.4), 262.0 (α-OH-Δ4-Δ8- GlcCer at m/z 742.4), 576.4 (α-OH-Δ4-Δ8,9methyl-ceramide at m/z 594.4), and 562.4 (α-OH-Δ4-Δ8-ceramide at m/z 580.4).